This invention relates to methods of synthesizing oligonucleotides and derivatives thereof on solid supports.
Oligonucleotides and derivatives thereof are typically prepared through solid phase synthesis regimes performed on solid supports packed in flow-through reactors. Flow-through reactors deliver solvent and reagent to solid supports at a required flow rate. There is an inherent resistance, or back pressure, associated with delivering solvent and or reagent to the solid support. The resistance can be caused by the solvent, the reagent lines, and/or the solid support in the reactor. Back pressure is undesirable because it distorts reaction conditions and makes synthesis techniques less efficient.
The back pressure caused by the solid support depends on the chemical composition of the support. Back pressure is also affected by the physical-chemical characteristics of the support and the packing in the flow through reactor.
The required flow rate of solvents and reagents through a reaction vessel is assured by pumps that overcome back pressure. As back pressure increases, more pumping power is required and modifications to reaction conditions may be necessary. However, pumping power requirements can limit reaction vessel""s size or flow-through capacity, and the quality of oligonucleotide synthesized can also be degraded by unfavorable pumping characteristics.
In addition, as oligonucleotide synthesis progresses back pressure increases. If steps are not taken to control the increase in back-pressure before allowed pressure maxima are exceeded, the synthesis cycle has to be modified or otherwise the reaction vessel could become xe2x80x9ccloggedxe2x80x9d. Such modifications can include adjusting the flow rate through the reaction vessel or decreasing the size of the reactor.
Low flow rates and smaller reactors are undesirable because of the lower concentration and yields of oligonucleotides that can be synthesized. Similarly, flowrate adjustments during synthesis are undesirable because they are time consuming and may result in inferior oligonucleotide products. Hence, methods which address these needs have long been sought. This invention is directed to these, as well as other, important ends.
In some embodiments, the invention provides methods for synthesizing an oligomeric compound on a solid support comprising contacting a solid support with a filling material to produce a mixture thereof; placing the mixture in a reaction vessel suitable for use in solid phase synthesis; and synthesizing the oligomeric compound.
In further embodiments, the invention provides reaction vessels for synthesizing oligomeric compounds comprising a solid support; and a filling material.
In further embodiments, the invention provides apparatuses for synthesizing oligomeric compounds comprising a reagent source, and a reaction vessel, said reaction vessel comprising a solid support; and a filling material.
In further embodiments, the invention provides compositions comprising a solid support suitable for oligonucleotide synthesis and a filling material mixed therewith.
In some preferred embodiments of the foregoing methods, apparatuses and compositions, the filling material is glass, preferably glass beads. In further preferred embodiments of the foregoing methods, apparatuses and compositions, the filling material is a polymer, preferably polymeric beads.
In some preferred embodiments of the methods of the invention, the contacting and placing steps occur simultaneously in the reaction vessel. In further preferred embodiments, the oligomeric compound is an oligonucletide, preferably synthesized using phosphoramidite chemistry.
In some preferred embodiments, the filler material is glass, preferably glass beads, and the solid support is a polymer or glass beads, preferably Primer(trademark) 200 support, Tentagel support or POROS support.
In some preferred embodiments, the methods of the invention are used to prepare oligomeric compounds that are oligoncucleotides containing at least one phosphorothioate internucleoside linkage.